Following complete spinal cord injury (SCI), bladder contractions relying solely on spinal cord pathways emerge but neurogenic voiding commonly ensues: bladder contractions are often inappropriately frequent (hyperreflexia) and occur together with contraction of the EUS, preventing effective voiding (bladder-sphincter dyssynergia). Understanding the changes in reflex pathways is vital if effective therapies are to be developed for the debilitating consequences of SCI on the lower urinary tract. Our overall goal is to use a combination of neurotoxin treatment, pharmacology, and microanatomy to define the changes in spinal cord circuitry subserving micturition reflexes in rats with SCI. Hypotheses: Our first hypothesis is that TRPV1-/IB4- tachykininergic bladder afferents terminating adjacent to NK1R+ micturition-related neurons in SCI rats are responsible for the phasic voiding contractions (phVCs; spinal micturition reflex plus phasic EUS reflex) whereas TRPV1+/IB4+ tachykininergic bladder afferents terminating adjacent to NK1R+ micturition-related neurons are responsible for dyssynergic nonphasic voiding contractions (npVCs; spinal micturition reflex with only tonic EUS reflex). Our second hypothesis is that the targets of these tachykininergic afferents remain former projection neurons, but that these have acquired new spinal targets effecting the spinal micturition reflexes. Our third hypothesis is that these projection neurons and other spinal neurons involved in the spinal micturition reflex undergo central sensitization so that they become responsive to preemptive treatment against NMDA receptors, NK1 receptors, and reactive oxygen species. The specific objectives to attain these goals are: Specific objective 1. Discover the nature and spinal cord targets of bladder afferent neurons responsible for micturition reflexes after SCI. Our strategy is to ablate different subpopulations of afferent neurons, determine the effect of ablation on spinal micturition reflexes, and characterize changes in relationships between afferent neurons and their spinal cord targets. Specific objective 2. Localize, quantify, and determine the phenotypes of micturition-related ascending spinal projection neurons (ASPNs) connecting to brainstem and lumbar spinal cord targets in spinally intact animals and define the effects of lesions to individual populations of ASPNs on micturition in spinally intact and spinal cord injured animals. Our strategy is to characterize different populations of micturition-related ASPNs by retrograde transport of CTB from brainstem nuclei and determine the effect of their ablation by suicide transport of CTB-saporin. Specific objective 3. Determine the pharmacological and morphological phenotypes of spinal cord neurons involved in the spinal micturition reflex. Our strategy is to determine whether inhibitors of central sensitization-associated elements (favorably) affect micturition reflexes, whether such inhibitors can preemptively reduce or eliminate signs of central sensitization, and to localize such elements in fos-expressing micturition-related neurons. PUBLIC HEALTH RELEVANCE: Relevance of the proposed research to veterans health and/or healthcare issues: Some 250,000 people in the US have spinal cord injury with another 10,000 sustaining spinal cord injury every year. About 40,000 are military veterans, and of those about 60% incurred their SCI during military service in OEF/OIF or otherwise. Incontinence and bladder-sphincter dyssynergia (bladder and sphincter contracting at the same time so that voiding is only partial) are extremely common in spinal cord injured patients and very seriously degrade their quality of life. Understanding of the changes within the spinal cord promoting lower urinary tract dysfunction is a prerequisite for design of effective therapies.